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ES Cell Electroporation

We perform gene targeting in the murine genome by electroporation of pluripotent stem cells with targeting vectors, to achieve the introduction of known mutations in a pre-determined locus.

You will provide us with the targeting vector amplified and linearized (at least 100-120 ug). We will take over the last step of purification and quantification, prior to electroporating it. After electroporation we select the cells according to the resistance cassette introduced in the targeting vector (it is beneficial to introduce a negative selection cassette as well). After selection we pick and culture around 300 individual colonies, if possible, and triplicate them. We freeze down two of the triplicates and hand the third over the user, to prepare DNA for the screening of recombinant clones. Once recombination of both homology arms is clearly detected, we thaw and expand three positive clones for subsequent karyotyping and embryo injection. It is highly recommendable to test the screening strategy before electroporating the vector.

By default, we generate electroporate hybrid 129;C57Bl/6 ES cells, however other backgrounds can be considered upon request.